Small RNAs (sRNAs) regulate growth and development and maintain genome integrity through their control of gene expression and silencing of transposable elements (TEs). We hypothesized that investigating changes in sRNA accumulation following hybridization in maize would provide insight into mechanisms contributing to hybrid vigor in plants. We used Illumina sequencing to assess how sRNA populations vary between two maize inbred lines (B73, Mo17) and their vigorous hybrid (B73 x Mo17). We sampled sRNAs from the seedling shoot apex and the developing ear, two rapidly growing tissues that program the greater growth of maize hybrids.

In plants, sRNAs can be grouped into three functionally distinct classes based on their lengths of 21, 22, or 24 nucleotides (nt), and our studies provided insights into how the activities of each of these classes are impacted by hybridization. MicroRNAs (miRNA) are typically 21-nt and often regulate plant growth, but we found that hybridization does not significantly alter miRNA accumulation. One miRNA that does show genotypic variation is microRNA172 (miR172), and its activity is reduced in transgenic maize plants that over-express glossy15 (gl15). Reducing miR172 activity in maize slows growth rate and affects harvest index by delaying shoot maturation and flowering time. We found that altering the balance of gl15 and miR172 affects the degree of hybrid vigor for reproductive and vegetative growth for three crosses, suggesting that miR172 activity contributes to heterosis in maize. The most abundant class of sRNAs in maize are the 24-nt small interfering RNAs (siRNAs) that silence abundantly repeated sequences such as TEs. In contrast to miRNAs and as observed previously in both Arabidopsis and rice, hybridization passes on parental differences in siRNA populations, and for those 24-nt siRNAs that do differ between parents, there is a trend toward downregulation following hybridization. Surprisingly, hybrid vigor for B73xMo17 is fully maintained when 24-nt siRNAs are globally reduced by mutation of the RNA-dependent RNA polymerase2 (RDR2), which is encoded by modifier of paramutation1 (mop1). However, both the degree of inbreeding depression and phenotypic variation are greater in F2 and F3 populations derived from the B73xMo17 mop1 hybrids, suggesting that mop1 mitigates the genetic stress of inbreeding and that the silencing of genes and TEs by 24-nt siRNAs may contribute to trans-generational inheritance. The third class of small RNAs in maize is 21-22-nt siRNAs associated with the activity of a number of distinct long terminal repeat (LTR)-retrotransposon families. These 21-22-nt siRNAs differentially accumulate between B73 and Mo17 as well as their hybrid.

We extended this analysis of sRNA families to 36 diverse inbred lines. We found that high copy number families produce the most siRNAs and have activity in all genotypes but at variable levels; whereas, less active families are more likely to show strong differences in the presence or absence of siRNAs in specific genotypes. Overall, the accumulation of 21-22-nt siRNAs is more variable across maize LTR-retrotransposon families than 23-24-nt siRNAs. Genetic variation for LTR-siRNAs is not strongly correlated with the genomic copy number or distribution of families, and is also distinct from the patterns of DNA genetic variation. Within one breeding cycle, DNA and LTR-siRNA variation can change in similar or dissimilar directions, and divergent selection over many cycles can produce lines with different activities of families. We also discovered that divergence of LTR-siRNA profiles is prominent among genotypes representing germplasm groups that have been artificially isolated to exploit hybrid vigor. These results indicate that LTR-siRNAs contribute another component to regulatory diversity in complex genomes, which has the potential to regulate both TEs and genes at a genomic scale. The greater diversity that maize possesses in this regulatory variation may contribute to the species' high degree of heterosis and the ability of plant breeders to successfully harness it.